“Intraclonal Diversification and Evolution in Chronic Lymphocytic Leukemia Patients by High-Throughput Sequencing of IGHV-D-J Rearrangements” was a talk presented during this session by Davide Bagnara from the University of Genova, Italy.

It has been previously reported in Sanger sequencing studies than IGHV-D-J of CLL clones can undergo intra-clonal heterogeneity.

This group performed high-throughput sequencing on the IGHV-D-J repertoire of FACS sorted CD19+CD5+ cells from 39 patients with treatment naïve CLL. From mRNA, and using a set of primers covering every IGHV gene, full-length IGHV-D-J repertoire was amplified. Unique Molecular Identifiers (for error) and Polymerase Chain Reaction (for correction) were used to prepare libraries. The CD19+CD5+ compartment in patients with CLL contains both leukemic and non-leukemia B-cell clones.

In a group of M-CLL cases, it appeared that the CLL clone evolved from a precursor differing in IGHV mutational status

Two groups identified:

Almost all subclones derive directly from the CLL clone

A number of subclones develop in a complex, branching, phylogenetic tree; not directly deriving from one CLL clone

When analyzing the change in proportion of a clone at two different time points:

An increase in CLL clone indicates it has gained an advantage over the other subclones, not present in a previous state

A decrease in CLL clone indicated one or more subclones numerically competed with it, indicating ongoing clonal diversification

It was found that CD19+CD5+ non-leukemic B-cells could be oligoclonally expanded

CLL presents stereotypical IGHV-D-J sequences that can often be observed in clones of the non-leukemia CD19+CD5+ compartment

In conclusion, CLL clones diversify in vivo, acquiring IGHV-D-J mutations resulting in a level of clonal complexity not fully comprehended thus far, and takes place in both U- and M-CLL. The group hypothesized that the ongoing evolution of IGHV-D-J is likely due to Activation Induced Deaminase. Moreover, they suggested that ongoing IGHV-D-J could be used as a marker of DNA changes taking place across the genome and so presents as a measure of genomic instability.